The ability to move DNA from one cell to another is a powerful tool in modern molecular biology, yet the idea that this movement might be possible predates the current revolution in genetic engineering. In 1928, Griffith paved the way for the discovery that nucleic acids are the genetic material when he noticed that the virulence of bacteria could be altered by mixing live bacteria with solutions derived from killed bacteria. By the early 1960's, not only was the structure of the relevant component of the solution, DNA, solved, but it was already established that DNA could be moved into mammalian cells (Syzbalski, 1961). The focus of these early days of molecular biology and tissue culture were irreversibly changed by two critical developments: the discovery of calcium phosphate precipitation, a simple procedure to introduce DNA into immortalized cells in culture (Graham and van der Eb, 1972) and the isolation and characterization of mammalian globin, insulin, and growth hormone genes in the mid- to-late 1970's.
Today, the ability to manipulate DNA and to introduce it into cells has profound practical implications for human health. Recombinant proteins produced by such manipulations are becoming widely accepted treatments for a number of human diseases and play major roles in agriculture. Though far less developed, the field of human gene therapy also has been and will continue to be influenced by improvements in technologies for the manipulation of DNA.
Gene therapy is a medical intervention in which a small number of the patient's cells are modified genetically to treat or cure any condition, regardless of etiology, that will be ameliorated by the long-term delivery of a therapeutic protein. Gene therapy can, therefore, be thought of as an in vivo protein production and delivery system, and almost all diseases that are currently treated by the administration of proteins (as well as several diseases for which no treatment is currently available), are candidates for treatment using gene therapy. The field can be divided into two areas: germ cell and somatic cell gene therapy. Germ cell gene therapy refers to the modification of sperm cells, egg cells, zygotes or early stage embryos. On the basis of both ethical and practical criteria, germ cell gene therapy is inappropriate for human use. From an ethical perspective, modifying the germ line would change not only the patient, but also the patient's offspring and, to a small but significant extent, the human gene pool as a whole.
In contrast to germ cell gene therapy, somatic cell gene therapy would affect only the person under treatment (somatic cells are cells that are not capable of developing into whole individuals and include all of the body's cells with the exception of the germ cells). As such, somatic cell gene therapy is a reasonable approach to the treatment and cure of certain disorders in human beings. In a somatic cell gene therapy system, somatic cells (i.e., fibroblasts, hepatocytes or endothelial cells) are removed from the patient, the cells are cultured in vitro, the gene(s) of therapeutic interest are added to the cells and the genetically-engineered cells are characterized and reintroduced into the patient. The means by which these five steps are carried out are the distinguishing features of a given gene therapy system.
To provide an overview of how somatic cell gene therapy might be applied in practice, an example concerning the treatment of hemophilia B will be considered. Hemophilia B is a bleeding disorder that is caused by a deficiency in Factor IX, a protein normally found in the blood. As a candidate for a gene therapy cure, an affected patient would have an appropriate tissue removed (i.e., bone marrow biopsy to recover hematopoietic stem cells, phlebotomy to obtain peripheral leukocytes, a liver biopsy to obtain hepatocytes or a punch biopsy to obtain fibroblasts or keratinocytes). The patient's cells would be isolated, genetically engineered to contain an additional Factor IX gene that directs production of the missing Factor IX and reintroduced into the patient. The patient is now capable of producing his or her own Factor IX and is no longer a Hemophiliac. The physician will most likely schedule close follow up in the weeks and months after the treatment, but in a literal sense, the patient would have been cured.
In state-of-the-art somatic cell gene therapy systems, it is not possible to direct or target the additional therapeutic DNA to a preselected site in the genome. In fact, in retrovirus-mediated gene therapy, the most widely utilized experimental system retroviruses integrate randomly into independent chromosomal sites in millions to billions of cells. This mixture of infected cells is problematic in two senses: first, since integration site plays a role in the function of the therapeutic DNA, each cell has a different level of function and, second, since the integration of DNA into the genome can trigger undesired events such as the generation of tumorigenic cells, the likelihood of such events is dramatically increased when millions to billions of independent integrations occur.
The problems of populations consisting of large numbers of independent integrants might be avoided in two ways. First, a single cell with a random integration site could be propagated until sufficient numbers of the cloned cell could be introduced into the individual. The cells that make up this clonal population would all function identically. In addition, only a single integration site would be present in the clonal population, significantly reducing the possibility of a deleterious event. Second, a single cell or a population of cells could be treated with therapeutic DNA such that the DNA sequences integrate into a preselected site in the genome. In this case, all the cells would be engineered identically and function identically. Furthermore, the risk of a deleterious integration event would be eliminated. Both the above solutions are demonstrated in this application.
The application of targeting to somatic cell gene therapy has several other advantages in addition to simply introducing additional genes or functional DNA sequences into a cell. In targeted gene therapy, it would be possible to repair, alter, replace or delete DNA sequences within the cell. In the illustration of somatic cell gene therapy discussed above, for example, targeting would allow the patient's non-functional Factor IX gene to be repaired. The ability to repair, alter, replace and delete DNA sequences utilizing targeting technology would expand the range of diseases suitable for treatment using gene therapy (and for the in vitro production of recombinant proteins as well). As the above discussion suggests, it would be extremely useful to be able to target primary and secondary vertebrate cells.